Cell signaling involves the concerted activity of multiple second messenger pathways. It is the balance of these different signaling components, coordinated in both space and time, that ultimately dictate the response of the cell. While this is well understood in theory, the practice of measuring signaling is often reduced to two time points—before and after drug—and to a single second messenger. When kinetic measurements of signaling are possible, a new level of precision and insight guide new experiments and optimized assays. In the cases that it has been possible to image multiple components of a signaling pathway in the same cells (1-5), the interplay between the different components has provided new insights into the biological system and the downstream consequences of a drug's actions.
Multiplex sensors capable of simultaneously detecting different signaling components serve an important role in understanding complex biological pathways and assessing the biological relevance of a particular drug (8). For example, many drugs act at G-protein coupled receptors on the cell surface. Some of these receptors couple to the heterotrimeric protein, Gq, which activates phospholipase C (PLC). PLC in turn cleave PIP2 to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). This coordinated increase, in both DAG and cytosolic Ca2+, triggers the activation of conventional isoforms of protein kinase C (cPKC) which then phosphorylate many different targets. To unambiguously resolve PLC pathway activation, and to better understand the kinetics of these coordinated, parallel signaling processes and their significance in health and disease, multiplex sensor systems are needed that can simultaneously measure multiple molecules such as DAG, PIP2 and Ca2+.
Optimal multiplex sensors must satisfy a number of criteria. First, they must be capable of working in living cells and provide kinetic data for each signaling pathway. This means they need to work in living cells and provide strong signals that can be sampled at 10 Hz. Additionally, each sensor needs to consume as little of the visible spectrum as possible so that there is minimal crosstalk with other sensors. Furthermore, each sensor has to specifically detect the analyte at physiologically relevant concentrations.
Fluorescent protein-based sensors meet many of the design criteria: they work in living cells, they produce strong signals that can be sampled repeatedly and quickly, and the protein domains they carry have evolved to specifically detect a particular second messenger (1). However, early sensors based upon Forster Resonance Energy Transfer (FRET) between two different fluorescent proteins, rarely produce the sort of robust signals necessary for automated detection. Furthermore, the broad absorption bands of the donor and acceptor fluorophores consume most of the visible spectrum (12, 13).
More recently, a new generation of fluorescent protein sensors has been developed that only uses one fluorescent protein, produces large changes in fluorescence, and has the potential for multiplexing. Many of these new sensors carry a single, circularly permuted fluorescent protein that converts analyte binding into changes in fluorescence intensity. The green fluorescent GCaMP Ca2+ sensors (14-16), the red R-GECO1 Ca2+ sensor (17), the green ElectricPk voltage sensor (18), and the green cGMP sensor (19) use this approach. However, there continues to be a need in the art for additional novel fluorescent sensors that are robust, sensitive, can detect specific analytes and can be used in multiplex systems in real time and in relevant tissues and cell types. The invention of the present application addresses such need.